U.S. patent application number 11/672597 was filed with the patent office on 2007-09-13 for apparatus for photon detection.
Invention is credited to Takashi Anazawa, Tsuyoshi Sonehara.
Application Number | 20070210269 11/672597 |
Document ID | / |
Family ID | 38477998 |
Filed Date | 2007-09-13 |
United States Patent
Application |
20070210269 |
Kind Code |
A1 |
Sonehara; Tsuyoshi ; et
al. |
September 13, 2007 |
APPARATUS FOR PHOTON DETECTION
Abstract
The object of the present invention is to acquire the brightness
of NA>1 while alleviating the requirement for the precision of
positioning for the collection lens of the sample cell in a
non-liquid immersion system. In order to achieve the object
mentioned above, the bottom of the sample cell is formed in a
curved surface, and an arrangement is made to ensure that the
fluorescence irradiated from the focusing point would be parallel
pencils when emitted by the cell, and in addition a pinhole is
disposed at the focal point of the fluorescence collection
lens.
Inventors: |
Sonehara; Tsuyoshi;
(Kokubunji, JP) ; Anazawa; Takashi; (Koganei,
JP) |
Correspondence
Address: |
ANTONELLI, TERRY, STOUT & KRAUS, LLP
1300 NORTH SEVENTEENTH STREET, SUITE 1800
ARLINGTON
VA
22209-3873
US
|
Family ID: |
38477998 |
Appl. No.: |
11/672597 |
Filed: |
February 8, 2007 |
Current U.S.
Class: |
250/576 |
Current CPC
Class: |
G01N 21/6428 20130101;
G01N 21/0303 20130101; G01N 2021/6482 20130101; G01N 2021/6463
20130101; G01N 21/6452 20130101 |
Class at
Publication: |
250/576 |
International
Class: |
G01N 21/85 20060101
G01N021/85; G01N 15/06 20060101 G01N015/06; G01N 21/49 20060101
G01N021/49 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 13, 2006 |
JP |
2006-067937 |
Claims
1. An apparatus for photon detection comprising: a sample cell of
which the outer surface of the bottom is curved; a photodetector
for detecting the light irradiated from the bottom of said sample
cell; a pinhole plate located between said sample cell and said
photodetector; and a collection lens located between said sample
cell and said pinhole plate, where said pinhole plate is fixed in
such a way that the position of the pinhole created on said pinhole
plate may coincide with the focus of said collection lens.
2. The apparatus for photon detection according to claim 1,
comprising a light irradiating unit for irradiating said bottom of
said sample cell with excitation beams substantially as collimated
beams.
3. The apparatus for photon detection according to claim 2, wherein
said exciting beams are focused in a pinpoint in the sample
solution contained in said sample cell by the bottom of said sample
cell.
4. The apparatus for photon detection according to claim 2, wherein
a dichroic mirror is located between said sample cell and said
collection lens, and said light irradiating unit irradiates the
sample solution contained in said sample cell with excitation beams
through said dichroic mirror.
5. The apparatus for photon detection according to claim 1, wherein
the bottom of said sample cell has a refractive index of 1.55 or
more.
6. The apparatus for photon detection according to claim 1, wherein
a transparent partition is provided between said sample cell and
said collection lens.
7. The apparatus for photon detection according to claim 1, wherein
the outer surface of the bottom of said sample cell is
aspheric.
8. The apparatus for photon detection according to claim 7, wherein
the inner surface of the bottom of said sample cell is
aspheric.
9. The apparatus for photon detection according to claim 1, wherein
said sample cell has a cover made of a transparent or partially
light-absorbing material and at least a part of the lower side of
the cover is in contact with the sample solution contained in said
sample cell.
10. The apparatus for photon detection according to claim 1,
wherein said sample cell has a plurality of wells containing
respectively sample solution independently and a driving unit for
driving said sample cell two-dimensionally with reference to said
apparatus for photon detection.
11. An apparatus for photon detection comprising: a sample cell of
which the bottom of the outer surface is curved; a photodetector
for detecting the light irradiated from the bottom of said sample
cell; and a collection lens located between said sample cell and
said photodetector, wherein said photodetector is located at such a
position where the focus of said collection lens may coincide with
the photosensitive area.
12. The apparatus for photon detection according to claim 11,
comprising a light irradiating unit for irradiating said bottom of
said sample cell with excitation beams substantially as collimated
beams.
13. The apparatus for photon detection according to claim 12,
wherein said exciting beams are focused in a pinpoint in the sample
solution contained in said sample cell by the bottom of said sample
cell.
14. The apparatus for photon detection according to claim 12,
wherein a dichroic mirror is located between said sample cell and
said collection lens, and said light irradiating unit irradiates
the sample solution contained in said sample cell with excitation
beams through said dichroic mirror.
15. The apparatus for photon detection according to claim 11,
wherein the bottom of said sample cell has a refractive index of
1.55 or more.
16. The apparatus for photon detection according to claim 11,
wherein a transparent partition is provided between said sample
cell and said collection lens.
17. The apparatus for photon detection according to claim 11,
wherein the outer surface of the bottom of said sample cell is
aspheric.
18. The apparatus for photon detection according to claim 17,
wherein the inner surface of the bottom of said sample cell is
aspheric.
19. The apparatus for photon detection according to claim 11,
wherein said sample cell has a cover made of a transparent or
partially light-absorbing material and at least a part of the lower
side of the cover is in contact with the sample solution contained
in said sample cell.
20. The apparatus for photon detection according to claim 11,
wherein said sample cell has a plurality of wells containing
respectively sample solution independently and a driving unit for
driving said sample cell two-dimensionally with reference to said
photodetector.
Description
CLAIM OF PRIORITY
[0001] The present application claims priority from Japanese
application JP 2006-067937 filed on Mar. 13, 2006, the content of
which is hereby incorporated by reference into this
application.
FIELD OF THE INVENTION
[0002] The present invention relates to an apparatus for photon
detection preferred in quantifying fluorescence-labeled
biomolecules by irradiating sample solution containing the
biomolecules with photon and by detecting the excited
fluorescence.
BACKGROUND OF THE INVENTION
[0003] To quantify fluorescence-labeled biomolecules, an analysis
apparatus for irradiating the sample solution with photon in such a
way that photons may converge at a point (hereinafter referred to
as "focusing point") within the solution to excite the
fluorescence, collecting the fluorescence irradiated by the
biomolecules in the solution and transmitting through the sample
cell with the collection lens, and thus detecting only the photons
forming image on a pinhole placed on the conjugate point of the
focusing point and transmitting through the pinhole is generally
used. By detecting only the photons transmitting through the
pinhole, the volume for detecting the fluorescence in the sample
(detection volume) is limited to the vicinity of the focusing
point, and background light (B) such as luminescence of solution at
anywhere other than the vicinity of the focusing point, scattering
light at the surface of the sample cell is blocked, and thus it is
possible to detect the fluorescence from biomolecules in the
detection volume (signal, S) with a good signal to background (S/B)
ratio.
[0004] The detection system of this type of apparatus is generally
constituted as shown in FIG. 1. Excited beam is focused at the
sample 1 in the sample cell 2, and the fluorescence 4 emitted from
the focusing point 3 is collected at the pinhole on the pinhole
plate 6 created at a conjugate position with the focusing point 3
by means of the collection lens 5, and is detected by the
photodetector 7 passing through the pinhole. In reality, the lens 5
is a combination lens made of a plurality of lens, incorporating a
filter, a dichroic mirror and the like between the lens. In FIG. 1
which is a schematic illustration showing the basic principle,
however, we used a simplified structure for its illustration. As B
is reduced to a sufficiently low level in a system like the one
shown in FIG. 1, the signal to noise (S/N) ratio of fluorescence
detection is proportionate to S. Therefore, it is necessary to
increase the value of S itself in order to increase the S/N ratio
value. There are two means for increasing the value of S. One is to
increase the intensity of photon being irradiated. And the other is
to increase the efficiency of fluorescence collection. Any
irradiation of light excessively strong risks to destroy the
fluorescent substance labeling the biological specimen, and
therefore there is a limit to this method. Accordingly, the
efficiency of fluorescence collection of the detection system must
be increased to the maximum extent possible. In other words, the
numerical aperture (NA) of the detection system must be increased
to the maximum extent possible. In order to detect the fluorescence
from one molecule with a good S/N ratio, a NA larger than one is
preferable. However, in order to achieve NA>1 by using a sample
cell 2 having a generally flat bottom, the lens 5 must be immersed
in a liquid as described in JP-A No. 85443/2004. In other words,
the space between the lens 5 and the sample cell 2 must be filled
with a liquid. As the liquid-immersion system involves a step of
filling the space between the lens and the sample cell with a
liquid, its operability is somewhat lower than that of the dry
system. In case of analyzing a variety of samples while moving the
sample cell, the troublesomeness of operation is remarkable. And
this leads to a high risk of generating bubbles in the liquid with
which the cell is filled and of impeding the transmission of
fluorescence. Although there are systems wherein the process of
charging liquid or removing bubbles is automatized as shown in JP-A
No. 85443/2004, a rise in the cost of equipment is unavoidable, and
the troublesomeness of filling liquid and replacing sample cells
remains.
[0005] Although in some documents the focusing point in the sample
and the conjugate point are called "focal point," this is an
imprecise expression, and in the present specification we used the
term "focal point" only in the sense of "a point where parallel
pencils introduced to the lens from the sample side are focused in
the side opposite the sample side." In order to clarify this point,
in FIG. 1, we indicated fluorescence of virtual parallel pencils 4'
and focal point 8. Thus, according to the prior art, the focal
point 8 is not located in the pinhole.
[0006] As shown in Patent documents 2-5, it is possible to realize
NA>1 even in a non-liquid immersion system (dry system) by
creating a curved surface on the bottom of the sample cell. When
this is combined with a detection system with a pinhole as shown in
Patent documents 2-5 and FIG. 1, a system shown in FIG. 2 can be
created. Since the light coming out of the sample cell shown in
Patent documents 2-5 is divergent light, the pinhole of the pinhole
plate 6 is not located at the focusing point 8 of the lens 5 also
in the system of FIG. 2. According to this system, it is possible
to realize NA>1 in a dry system.
SUMMARY OF THE INVENTION
[0007] In the system shown in FIG. 2, the divergent light passing
through the sample cell 2 is detected and unless the central axis
of the sample cell 2 and the optical axis of the collection lens 5
agree very precisely and the distance between the sample cell 2 and
the collection lens 5 agrees very precisely to a predetermined
value, an aberration occurs, and the image on the pinhole at the
focusing point 3 becomes blurred. As a result, the efficiency of
detecting fluorescence falls down and at the same time the
efficiency of removing the background light by the pinhole falls
down, resulting in a decline of the S/N ratio. FIG. 3 shows how a
displacement of the central axis 9 of the sample cell 2 from the
optical axis 10 of the collection lens 5 resulted in a blurred
image at the focusing point 3. Although it is difficult to express
in the figure, a shift in the relative positions of the sample cell
2 and the collection lens 5 in the direction of the optical axis
produces a similar effect. Therefore, in a system as the one shown
in FIG. 2, it is necessary to control very precisely the relative
positions of the sample cell 2 and the collection lens 5. This not
only results in a higher cost, but as the sample cell 2 is an item
that must be replaced frequently, it is not practical to proceed to
a precise positioning every time the sample cell 2 is replaced.
[0008] Thus, in order to obtain a degree of brightness of NA>1
using the prior fluorescence detection system with a pinhole, it is
necessary either to fill the space between the collection lens and
the sample cell with a liquid or to position the sample cell very
precisely in relation to the lens. Both of these approaches are not
suitable for automated processing of a large number of samples.
[0009] In the present invention, a sample cell the outer surface of
the bottom of which is curved acting as a lens is used. Excitation
beams are irradiated from the bottom of the sample cell as
collimated beams and the beams focused at the focusing point in the
sample solution. The fluorescence generated by the irradiation of
the excitation beams are taken up as the collimated beams from the
bottom of the sample cell, are collected by the collection lens,
and are detected by the photodetector. At this time, the position
of the pinhole is adjusted to agree with the focusing point of the
collection lens, and a pinhole plate is installed between the
sample cell and the photodetector. Or, in stead of using a pinhole
plate, the photodetector is located at such a position where the
photosensitive area of the photodetector agrees with the focusing
point of the collection lens.
[0010] According to the present invention, NA>1 can be realized
in a dry system, and the effect of removing the background light
can be obtained without a high-precision positioning of the sample
cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is an illustration of the structure of a conventional
fluorescence detection system having a pinhole;
[0012] FIG. 2 is an illustration of the structure combining a
sample cell whose bottom is a curved surface and a conventional
fluorescence detection system having a pinhole;
[0013] FIG. 3 is an illustration of the case wherein the relative
position of the sample cell and the collection lens has shifted one
against the other in the conventional optical system;
[0014] FIG. 4 is an illustration of the structure of a detection
optical system according to the present invention;
[0015] FIG. 5 is an illustration of the case wherein the relative
position of the sample cell and the collection lens has shifted one
against the other in the detection system according to the present
invention;
[0016] FIG. 6 represents graphs showing the range of forms of
sample cell where NA is >1;
[0017] FIG. 7 is an illustration of the structure of the first
embodiment of the present invention;
[0018] FIG. 8 is an enlargement of the top plan view of the portion
above the cell stand 21;
[0019] FIG. 9 represents graphs showing the relationship between
the efficiency of fluorescence detection and the displacement of
the relative position of the collection lens and the sample cell in
the system of the present invention and the system shown in FIG.
2;
[0020] FIG. 10 is the front view and the sectional view of the cell
according to the second embodiment of the present invention;
[0021] FIG. 11 is the front view of the vicinity around the cell
according to the second embodiment of the present invention;
[0022] FIG. 12 is the sectional view of the cell according to the
fourth embodiment of the present invention;
[0023] FIG. 13 is an illustration describing the fabrication
process of the cell according to the fourth embodiment of the
present invention;
[0024] FIG. 14 is the sectional view of the vicinity around the
cell according to the fifth embodiment of the present
invention;
[0025] FIG. 15 is the sectional view of the vicinity around the
cell according to the sixth embodiment of the present invention;
and
[0026] FIG. 16 is an illustration of the structure of the seventh
embodiment of the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0027] FIG. 4 is an illustration showing the structure of the
detection system unit of the apparatus for photon detection
according to the present invention. In the present invention, as
shown in FIG. 4, the bottom of the sample cell is curved so that
the fluorescence emitted from the focusing point 3 may constitute
parallel pencils when they are irradiated from the sample cell, and
the pinhole is created at the focusing point 8 of the fluorescence
collection lens 5.
[0028] FIG. 5 is a schematic illustration of the detection system
shown in FIG. 4 in which the relative positions of the sample cell
and the collection lens have shifted in the vertical direction of
the optical axis from the best positions. As a result of thus
creating parallel pencils between the sample cell and the
collection lens, no aberration occurs even in case of a
displacement of both of them, and a high ratio of fluorescence
passing through the pinhole is maintained.
[0029] The representative materials of the member through which
photons are allowed to transmit are quartz, BK7, and acrylic
plastic, and the refractive index of these materials is
approximately 1.5 (to be more precise 1.46-1.54). The lens for
optical disks and the like are constituted by the materials
mentioned above. On the other hand, as the inner side of the sample
cell according to the present invention is in contact with a liquid
having a refractive index of 1.3-1.4, it is expected that, in view
of the refractive index of 1.5, the refractive power of the inner
surface would be insufficient. Accordingly, we carried out a
thorough analysis on the refractive index of the materials and the
achievable NA based on a beam pursuit simulation. In the following
description, the refractive index of the cell materials is shown by
n.
[0030] As is well known, the form of making beams from one point
parallel materially without aberration is primarily determined only
by three parameters: the radius of curvature R1 at the top of the
outer surface, the focal distance f when the sample cell is
considered as a type of lens system, and the thickness t1 on the
central axis. When similar forms are considered as the identical
form, independent parameters representing the form of the sample
cell are limited to the standardized radius of curvature (R1/f) and
the standardized thickness (t1/f). In other words, the form of the
sample cell is represented by the combination of these two values
(R1/f, t1/f). And when the angle .theta. formed by the tangential
plane of the outer surface of the sample cell and the plane
orthogonal with the central axis of the cell becomes larger than
the Brewster's angle arctan (n), the reflectance of fluorescence on
the outer surface of the cell increases sharply, and the beams
cease to transmit effectively. Accordingly, we calculated by a
simulation the range of the form of the cell by which NA will be
>1 while meeting with the requirement of .theta.<arctan(n).
The result obtained is shown in FIG. 6. The range painted
completely in black is the range where NA is >1. Thus, as long
as n=1.5, there is no range where NA is >1, and n must be at
least n>1.55 to obtain NA>1, and it is preferable that
n>1.6 to obtain NA>1 in a wider range. Therefore, a
refractive index of 1.55 or more is required as the requirement for
the material of the sample cell according to the present invention.
And preferably the refractive index should be 1.6 or more, and more
preferably the same should be 1.7 or more.
[0031] The apparatus for photon detection according to the present
invention can be used as a low-cost and convenient fluorescence
correlation spectrometer, a high-sensitivity fluorescence plate
reader, a flow site meter and the like.
[0032] We will describe below specifically the embodiments of the
present invention with reference to drawings.
First Embodiment
[0033] FIG. 7 is an illustration of the structure of the first
embodiment of the apparatus for photo detection according to the
present invention. The light source 11 is a laser with a wavelength
of 532 nm, and an output of 1 mW. The parallel excitation beams 12
emitted by the light source 11, after having enhanced their
spectrum purity while passing through an excitation filter 13, are
reflected by a dichroic mirror 14, are irradiated onto the bottom
of the sample cell 2 as they remain parallel pencils to be focused
at the focusing point 3 in the sample 1. The fluorescence 4 emitted
from the biomolecules existing at the focusing point 3 by the
irradiation of the excitation beams is emitted from the bottom of
the cell 2 as parallel pencils due to the refraction at the
boundary surface of the cell, transmits the dichroic mirror 14,
shields the components having wavelength other than the
fluorescence wavelength such as the scattered light of the
excitation beams of 523 nm with a fluorescence filter 15, and is
focused into the pinhole created on the pinhole plate 6 by the
collection lens 5. The pinhole is located at the position of the
focal point 8 of the collection lens 5. The pinhole shields the
luminescence from parts other than the focusing point in the sample
and the sample cell, and only the fluorescence from the focusing
point passes through the pinhole and is detected by the
photodetector 7. The NA of this detection system is 1.15, the
diameter of the excitation beam at the focusing point is 1 .mu.m,
the image magnification of the collection lens 5 is 10 times, and
the diameter of the pinhole is 10 .mu.m. The solvent of the sample
is SSC buffer with a refractive index of 1.351. The material of the
sample cell is resin with a refractive index of 1.7. The space
between the sample cell and the collection lens is occupied by air,
and the creation of a curved surface on the bottom of the
collection lens thus enabled us to realize a NA larger than 1 in
the dry system.
[0034] The outer surface and the inner surface of the sample cell 2
are respectively aspheric as shown by the following equation.
z = cr 2 1 + 1 - ( 1 + K ) c 2 r 2 + Ar 4 + Br 6 + Cr 8 + Dr 10 +
Er 12 + Fr 14 + Gr 16 + Hr 18 [ Equation 1 ] ##EQU00001##
[0035] In this equation, r= (x.sup.2+y.sup.2), c is the curvature
at the top, K is the conic constant, and A, B, C, D, E, F, G, H are
aspheric coefficients The conic constant and the aspheric
coefficient representing the outer surface and the inner surface of
the sample cell 2 in the present embodiment and the thickness in
the central axis are shown in Table 1. The unit of r and z is mm.
By making both surfaces aspheric, it is possible to convert the
fluorescence from the focusing point into parallel pencils with a
resolution at the diffraction limit of wave aberration of 0.07
.lamda.rms or less. The NA for detection obtained by the present
embodiment is 1.15.
TABLE-US-00001 TABLE 1 Outer surface (air Inner surface (sample
side) solution side) Refractive 1.7 1.351 (Sample refractive index
index) Thickness on 3.88 0.54286 the central (Distance with the
axis focusing point) C 0.506894 1.7370 K -3.9 -6.49 A 0.5680637E-1
0.3759345 B -0.160145E-1 -0.9478041 C 0.6405858E-2 1.61106 D
0.2005395E-2 1.805153 E 0.4605048E-3 1.298703 F -0.68272E-4
-0.5743903 G 0.5808605E-5 0.1417377 H 0.2099961E-6 0.1490973E-1
[0036] The light source 11, the photodetector 7 and optics for
transmitting or reflecting beams are fixed inside the structure 20.
A hole is perforated in the structure 20 to allow the passage of
beams between the sample cell 2 and the dichroic mirror 14, and
this hole is covered with a transparent window plate 16. The
fixation of this window plate has the effect of preventing samples
flowing out of the sample cell and dusts contained in the
atmosphere from sticking on the optics below the dichroic
mirror.
[0037] The sample cell 2 is set on the cell table 21 fixed on the
structure 20. And the sample cell 2 is perforated to create
positioning holes 23-1 and 23-2. By placing the sample cell 2 in
such a way that positioning pins 22-1 and 22-2 fixed on the cell
table 21 may fit in these positioning holes, the sample cell 2 is
automatically positioned in such a way that the distance between
the optical axis of the fluorescence 4 and the excitation beams 12
and the symmetry axis of the sample cell may be limited to 0.1 mm
or less. FIG. 8 is a top plan view of the section above the cell
table 21. Thus, the positioning holes 23-1 and 23-2 of the sample
cell 2 are fitted respectively with the positioning pins 22-1 and
22-2.
[0038] FIG. 9 represents graphs showing the relationship between
the divergence of the cell position from the reference position in
the present embodiment and in the system resulting from the
replacement of the detection system of the present embodiment by
the conventional detection system shown in FIG. 2 and the
efficiency of detecting fluorescence from biomolecules. In this
case, both the inner surface and the outer surface of the sample
cell are spheric, and the sample cell has almost the same NA of
1.14 as the first embodiment. .DELTA.y represents a shift from the
ideal position of the sample cell in the direction of crossing
vertically with the optical axis of the detection system while
.DELTA.z represents a shift towards the optical axis. As shown in
FIG. 9, while a mere displacement of the sample cell by 10 .mu.m in
a system as the one shown in FIG. 2 leads to a sharp decline in the
efficiency of detecting fluorescence, according to this invention,
even if the position of the sample cell is displaced by 0.1 mm, the
efficiency remains virtually unchanged.
[0039] As described above, according to the present invention, it
is possible to realize NA>1 by the dry system. In addition, a
system of fluorescence detection wherein the efficiency of
detection does not practically fall down even if the position of
the sample cell is displaced. As a result, in the case of analyzing
a large number of samples by replacing sample cells, it will be no
longer necessary to adjust the position of fixing the sample cells,
and this will be very advantageous in practical use. And as the
precision of positioning sample cells is not severe, it will be
possible to continually analyze automatically a large number of
samples by driving plates carrying a large number of sample cells
by automatic parallel advancing stages. In particular, in the case
of driving plates at a constant speed, the time of moving over a
width of .+-.0.1 mm or more can be used for measurement according
to the present invention, while nothing other than the time
required for moving over a width of approximately 0.01 .mu.m can be
used for measuring a sample according to the system shown in FIG.
2. In other words, more than ten times the measuring time can be
taken and an improvement of S/N ratio of 10-3 or more can be
expected.
Second Embodiment
[0040] FIG. 10 represents a top plan view and cross sectional view
of the sample cell according to the second embodiment of the
present invention. According to the present embodiment, we created
a plurality of wells on the sample cell 2 so that a plurality of
samples 1-1 . . . 1-96 may be introduced therein, and we formed the
bottom of each well to constitute a curved surface so that the
similar effect as the first embodiment may be obtained from the
same. In the present embodiment, we disposed the wells at the 9 mm
pitch identical to the sample preparing plate generally used
arranged in 8 rows and 12 lines of wells per plate so that samples
may be transported easily by the sample dispensing robot available
on the market. Of course, a 4.5 mm pitch for 16 rows and 24 lines
of wells per plate can be adopted. Like in the first embodiment, we
created two positioning holes 23-1 and 23-2 in each sample cell
2.
[0041] The optical system of irradiating excitation beams and
detecting fluorescence in the present embodiment is basically same
as the first embodiment. FIG. 11 is an enlargement of the periphery
around a sample cell 2 fixed on an apparatus for photon detection.
In the present embodiment, the table 21 for accommodating a sample
cell 2 is fixed on an automatic xy stage 30, and the sample cell 2
is carried in the x and y directions. The precision of positioning
of the automatic xy stage 30 is 0.05 .mu.m, and the sample cell 2
is positioned and stands still so that the central axis of each
well may agree with the optical axis of the detection optical
system to this precision, and the fluorescence from these well is
detected thereby. By the computer control, the cycle of this
positioning and detection of fluorescence is repeated automatically
to each sample 1-1 . . . 1-96. Thus, in the present embodiment, the
action of setting once a sample cell 2 on the table 21 has the
effect of automatically analyzing 96 samples. Of course, if a
sample cell with 16 rows and 24 lines of wells is used, 384 samples
can be automatically analyzed. Needless to say, the number of rows
and lines of samples is not limited to the values mentioned
above.
Third Embodiment
[0042] The third embodiment of the present invention is constituted
almost in the same way as the second embodiment. However, the xy
stage 30 is not brought to a standstill every time when the axis of
each well and the optical axis of the detection system of
fluorescence agree, and is driven at almost at a fixed speed over a
row, and fluorescence is detected while the axis of the well and
the optical axis of the detection system agree within a range of
.+-.0.1 mm. With the conventional optical system, fluorescence
could be detected efficiently only while both axes agreed within a
range of .+-.0.01 mm. However, according to the optical system of
the present invention as shown in FIG. 9, fluorescence can be
detected efficiently while both axes agree within a range of
.+-.0.1 mm. As a result, it has become possible to detect
fluorescence during a length of time approximately ten times of the
conventional system, and it will be possible to detect fluorescence
with a sufficient S/N ratio even by such a continuous constant
speed driving. As it is no longer necessary to position the sample
cell 2 with a high precision according to the present embodiment,
no stepping motor is required as the motor for driving the
automatic xy stage, and as low-priced DC motor can be used to cope
with the situation, it will be possible to obtain the cost reducing
effect as a result.
Fourth Embodiment
[0043] FIG. 12 is a sectional view of a sample cell 2 according to
the fourth embodiment of the present invention. The form of the
sample cell 2 according to the present embodiment is almost the
same as the second embodiment. However, while the sample cell
according to the second embodiment was an integrally formed plastic
resin structure 2, the sample cell according to the present
embodiment is formed by gluing together two different members 2A
and 2B. FIG. 13 is an illustration showing the fabrication process
of the sample cell according to the present embodiment. A member 2A
wherein columnar through holes are arranged in an array is
positioned and glued together by means of an adhesive with another
member 2B where lenticules constituting the bottom of sample cells
on the lower surface are arranged in an array in the same way as
the member 2A.
[0044] As a result of fabricating the sample cells by gluing
together two members in this way, the form of each member is
simplified and the manufacturing cost has sharply decreased, and in
spite of the addition of a gluing process, we obtained the effect
of a reduction in the total manufacturing cost. Particularly the
member 2A being simply a flat substrate with through holes created
therein is not only easy to fabricate and unlike the member 2B
needs not to be transparent. Therefore, the margin of selecting the
material has expanded remarkably. The adoption of an opaque
material for the member 2B facilitates the shielding of outside
lights. We described here a sample cell having a plurality of
wells. However, it is possible to fabricate a sample cell having a
single container of sample as shown in FIG. 7 by gluing together
two members.
Fifth Embodiment
[0045] FIG. 14 is an enlargement of the sectional view of the
vicinity of the sample cell 2 according to the fifth embodiment of
the present invention. According to the present embodiment, the
sample cell 2 has a transparent cover 24, and a part of the lower
surface of the cover 24 is designed to come into contact with the
surface of the sample solution. As a result, the excitation beams
transmit upward through the sample cell without scattering at the
sample/air interface, and reduce the rise of the background light
caused by the scattered light at the surface of sample. As the
material of the cover 24, materials whose refractive index is close
to the sample can be preferably used. As the refractive index of
the material according to the present embodiment was 1.36, we used
an acrylic resin with a low refractive index (refractive index:
1.42). In addition, it is possible to reduce further scattering by
creating an anti-reflection coat on the upper surface (air/cover
interface) of the cover 24.
Sixth Embodiment
[0046] FIG. 15 is an enlargement of the sectional view of the
vicinity of the sample cell 2 according to the sixth embodiment of
the present invention. In the present embodiment, we created a
cover 24 on the sample cell 2 like the fifth embodiment. However,
we obtained the same effect as the fifth embodiment by adopting a
light-absorbing material instead of a transparent member. In the
present embodiment, we used a black polycarbonate as the material
of the cover 24. However, any material that absorbs light well may
do. We tried to improve the efficiency of absorption of light by
creating a conic depression 25 at a point where the light from the
cover 24 is irradiated, Even if the material of the cover itself
does not absorb light, it is possible to obtain the same effect by
applying a light-absorbing coating on the area irradiated by the
excitation beam.
Seventh Embodiment
[0047] FIG. 16 is an illustration showing the optical system of the
seventh embodiment of the present invention. We constituted the
present embodiment basically in the same way as the first
embodiment. However, we omitted the mechanical pinhole plate 6 by
arranging the photosensitive area 26 of the photodetector 7 in such
a way that the focusing point 8 of the collection lens 5 may be
contained therein. According to this structure, it is possible to
obtain the same effect of removing the background light as
disposing a gobo having a hole of a diameter equal to the effective
diameter of the photosensitive area at the focusing position
without pinhole. The peculiar effect of this structure is the
possibility of reducing a component part of the structure.
[0048] It is possible to apply respectively the structures
described in the embodiments 1-6 described above to the apparatus
for photon detection having the optical system of the present
embodiment.
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